Down-Regulation of GATA-3 Expression during Human Papillomavirus-Mediated Immortalization and Cervical Carcinogenesis
2002; Elsevier BV; Volume: 160; Issue: 6 Linguagem: Inglês
10.1016/s0002-9440(10)61143-1
ISSN1525-2191
AutoresRenske D.M. Steenbergen, Vanessa E. OudeEngberink, Debbie Kramer, Henri F.J. Schrijnemakers, René H.M. Verheijen, Chris J.L.M. Meijer, Peter J.F. Snijders,
Tópico(s)Epigenetics and DNA Methylation
ResumoTo identify cellular genes that may be involved in human papillomavirus (HPV)-mediated immortalization mRNA differential display analysis was performed on preimmortal and subsequent immortal stages of four human keratinocyte cell lines transformed by HPV type 16 or 18 DNA. This yielded a cDNA fragment encoding the transcription factor GATA-3 that was strongly reduced in intensity in all immortal stages of the four cell lines. A marked reduction in both GATA-3 mRNA and protein expression in HPV-immortalized cell lines was confirmed by reverse transcriptase-polymerase chain reaction, Western blot analysis, and immunohistochemistry and was also shown to be apparent in cervical carcinoma cell lines. Immunohistochemical analysis of cervical tissue specimens showed a clear nuclear staining for GATA-3 in normal cervical squamous epithelium (n = 14) and all cervical intraepithelial neoplasia (CIN) I (n = 6) and CIN II lesions (n = 2). In contrast, 11% (1 of 9) of CIN III lesions and 67% (8 of 12) of cervical squamous cell carcinomas revealed a complete absence of GATA-3 immunostaining. Hence, complete down-regulation of GATA-3 expression represents a rather late event during cervical carcinogenesis. Whether GATA-3 down-regulation is etiologically involved in HPV-mediated immortalization and cervical carcinogenesis remains to be examined. To identify cellular genes that may be involved in human papillomavirus (HPV)-mediated immortalization mRNA differential display analysis was performed on preimmortal and subsequent immortal stages of four human keratinocyte cell lines transformed by HPV type 16 or 18 DNA. This yielded a cDNA fragment encoding the transcription factor GATA-3 that was strongly reduced in intensity in all immortal stages of the four cell lines. A marked reduction in both GATA-3 mRNA and protein expression in HPV-immortalized cell lines was confirmed by reverse transcriptase-polymerase chain reaction, Western blot analysis, and immunohistochemistry and was also shown to be apparent in cervical carcinoma cell lines. Immunohistochemical analysis of cervical tissue specimens showed a clear nuclear staining for GATA-3 in normal cervical squamous epithelium (n = 14) and all cervical intraepithelial neoplasia (CIN) I (n = 6) and CIN II lesions (n = 2). In contrast, 11% (1 of 9) of CIN III lesions and 67% (8 of 12) of cervical squamous cell carcinomas revealed a complete absence of GATA-3 immunostaining. Hence, complete down-regulation of GATA-3 expression represents a rather late event during cervical carcinogenesis. Whether GATA-3 down-regulation is etiologically involved in HPV-mediated immortalization and cervical carcinogenesis remains to be examined. Infection with high-risk human papillomavirus (HPV) types is the most significant risk factor for the development of cervical cancer and HPV DNA can be detected in almost all cervical squamous cell carcinomas.1Walboomers JMM Jacobs MV Manos MM Bosch FX Kummer JA Shah KV Snijders PJF Peto J Meijer CJLM Munoz N Human papillomavirus is a necessary cause of invasive cervical cancer worldwide.J Pathol. 1999; 189: 12-19Crossref PubMed Scopus (6883) Google Scholar HPV functions are, however, not sufficient for the development of cervical cancer and additive oncogenic events involving host cell genes are required, consistent with a multistep process of carcinogenesis. To gain better insight in HPV-mediated carcinogenesis in vitro model systems have been proven very valuable. High-risk HPV types, in particular HPV 16 and HPV 18, can induce immortalization of primary human epithelial cells in vitro.2Dürst M Dzarlieva-Petrusevka T Boukamp P Fusenig NE Gissmann L Molecular and cytogenetic analysis of immortalized human primary keratinocytes obtained after transfection with human papillomavirus type 16 DNA.Oncogene. 1987; 1: 251-256PubMed Google Scholar, 3Pirisi L Yasumoto S Feller M Doniger J DiPaolo JA Transformation of human fibroblasts and keratinocytes with human papillomavirus type 16 DNA.J Virol. 1987; 61: 1061-1066Crossref PubMed Google Scholar, 4Steenbergen RDM Walboomers JMM Meijer CJLM van der Raaij-Helmer Parker JN Chow LT Broker TR Snijders PJF Transition of human papillomavirus type 16 and 18 transfected human foreskin keratinocytes towards immortality: activation of telomerase and allele losses at 3p, 10p, 11q and/or 18q.Oncogene. 1996; 13: 1249-1257PubMed Google Scholar Studies involving somatic cell fusions and microcell-mediated chromosome transfers have indicated that particularly recessive gene alterations are crucial for immortalization of HPV-containing epithelial cells.5Chen TM Pecoraro G Defendi V Genetic analysis of in vitro progression of human papillomavirus-transfected human cervical cells.Cancer Res. 1993; 53: 1167-1171PubMed Google Scholar, 6Steenbergen RDM Kramer D Meijer CJLM Walboomers JMM Trott DA Cuthbert AP Newbold RF Overkamp WJ Zdzienicka MZ Snijders PJF Telomerase suppression by chromosome 6 in a human papillomavirus type 16-immortalized keratinocyte cell line and in a cervical cancer cell line.J Natl Cancer Inst. 2001; 93: 865-872Crossref PubMed Scopus (63) Google Scholar HPV-mediated immortalization is associated with an arrest of telomeric shortening and activation of the telomere lengthening enzyme telomerase.4Steenbergen RDM Walboomers JMM Meijer CJLM van der Raaij-Helmer Parker JN Chow LT Broker TR Snijders PJF Transition of human papillomavirus type 16 and 18 transfected human foreskin keratinocytes towards immortality: activation of telomerase and allele losses at 3p, 10p, 11q and/or 18q.Oncogene. 1996; 13: 1249-1257PubMed Google Scholar, 7Klingelhutz AJ Barber SA Smith PP Dyer K McDougall JK Restoration of telomeres in human papillomavirus-immortalized human anogenital epithelial cells.Mol Cell Biol. 1994; 14: 961-969Crossref PubMed Scopus (176) Google Scholar Moreover, activation of telomerase, by up-regulation of its catalytic subunit human telomerase reverse transcriptase gene, seemed to be necessary and sufficient for immortalization of HPV-transfected keratinocytes.6Steenbergen RDM Kramer D Meijer CJLM Walboomers JMM Trott DA Cuthbert AP Newbold RF Overkamp WJ Zdzienicka MZ Snijders PJF Telomerase suppression by chromosome 6 in a human papillomavirus type 16-immortalized keratinocyte cell line and in a cervical cancer cell line.J Natl Cancer Inst. 2001; 93: 865-872Crossref PubMed Scopus (63) Google Scholar, 8Kiyono T Foster SA Koop JI McDougall JK Galloway DA Klingelhutz AJ Both Rb/p16INK4a inactivation and telomerase activity are required to immortalize human epithelial cells.Nature. 1998; 396: 84-88Crossref PubMed Scopus (1070) Google Scholar, 9Backsch C Wagenbach N Nonn M Leistritz S Stanbridge E Schneider A Durst M Microcell-mediated transfer of chromosome 4 into HeLa cells suppresses telomerase activity.Genes Chromosom Cancer. 2001; 31: 196-198Crossref PubMed Scopus (42) Google Scholar In addition, expression of the viral oncogene HPV 16 E6 from a heterologous promoter has been shown to activate telomerase in primary keratinocytes.10Klingelhutz AJ Foster SA McDougall JK Telomerase activation by the E6 gene product of human papillomavirus type 16.Nature. 1996; 380: 79-82Crossref PubMed Scopus (693) Google Scholar However, expression of HPV 16 and HPV 18 E6 from their native promoter in the context of the full-length virus genome did not result in telomerase activation,4Steenbergen RDM Walboomers JMM Meijer CJLM van der Raaij-Helmer Parker JN Chow LT Broker TR Snijders PJF Transition of human papillomavirus type 16 and 18 transfected human foreskin keratinocytes towards immortality: activation of telomerase and allele losses at 3p, 10p, 11q and/or 18q.Oncogene. 1996; 13: 1249-1257PubMed Google Scholar indicating that additive events are required. Recent studies have shown that yet unknown genes residing at chromosomes 3, 4, and 6 can regulate telomerase activity in immortal HPV-transformed cells.6Steenbergen RDM Kramer D Meijer CJLM Walboomers JMM Trott DA Cuthbert AP Newbold RF Overkamp WJ Zdzienicka MZ Snijders PJF Telomerase suppression by chromosome 6 in a human papillomavirus type 16-immortalized keratinocyte cell line and in a cervical cancer cell line.J Natl Cancer Inst. 2001; 93: 865-872Crossref PubMed Scopus (63) Google Scholar, 9Backsch C Wagenbach N Nonn M Leistritz S Stanbridge E Schneider A Durst M Microcell-mediated transfer of chromosome 4 into HeLa cells suppresses telomerase activity.Genes Chromosom Cancer. 2001; 31: 196-198Crossref PubMed Scopus (42) Google Scholar To identify host cell genes that may be involved in telomerase regulation and HPV-mediated immortalization we performed mRNA differential display analysis11Liang P Pardee AB Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction.Science. 1992; 257: 967-971Crossref PubMed Scopus (4675) Google Scholar on RNAs isolated from a panel of HPV-transformed keratinocytes at pre-immortal and subsequent immortal stages to identify genes showing differential expression. Two clonally derived HPV-16 transformed cell lines, designated FK16A and FK16B, as well as two clonally derived HPV 18-transformed cell lines, designated FK18A and FK18B, were analyzed. In all cell lines the acquisition of an immortal phenotype was well defined and shown to be associated with an arrest of telomere shortening and activation of telomerase.4Steenbergen RDM Walboomers JMM Meijer CJLM van der Raaij-Helmer Parker JN Chow LT Broker TR Snijders PJF Transition of human papillomavirus type 16 and 18 transfected human foreskin keratinocytes towards immortality: activation of telomerase and allele losses at 3p, 10p, 11q and/or 18q.Oncogene. 1996; 13: 1249-1257PubMed Google Scholar The cell lines FK16A, FK16B, FK18A, and FK18B were established by transfection of primary human foreskin keratinocytes (EK94-2) with the entire HPV 16 and HPV 18 genome, respectively, and cultured as described previously.4Steenbergen RDM Walboomers JMM Meijer CJLM van der Raaij-Helmer Parker JN Chow LT Broker TR Snijders PJF Transition of human papillomavirus type 16 and 18 transfected human foreskin keratinocytes towards immortality: activation of telomerase and allele losses at 3p, 10p, 11q and/or 18q.Oncogene. 1996; 13: 1249-1257PubMed Google Scholar The cervical carcinoma cell lines SiHa and HeLa were obtained from the American Type Culture Collection (Rockville, MD) and were cultured in Dulbecco’s modified Eagle’s medium (Life Technologies Inc., Breda, The Netherlands) supplemented with 10% fetal calf serum, penicillin (100 U/ml), streptomycin (100 μg/ml), and l-glutamine (2 mmol/L, Life Technologies). The breast carcinoma cell line MCF-7 was also obtained from the American Type Culture Collection and cultured in RPMI (Life Technologies Inc.) supplemented with 10% fetal calf serum, penicillin (100 U/ml), streptomycin (100 μg/ml), and l-glutamine (2 mmol/L, Life Technologies, Inc.). Of all cell lines cells were harvested by trypsinization at different passages before and after immortalization when the cells were growing exponentially. Four pre-immortal stages of cell lines FK18A and FK18B (FK18A p12, FK18A p13, FK18B p13, and FK18B p18; P = passage number) were compared with seven immortal stages of the different cell lines (FK16A p27, FK16A p30, FK16B p37, FK18A p25, FK18A p28, FK18B p27, and FK18B p61). Rat RNA was included as a control for aspecific bands. Total RNA was isolated using RNAzolB (Tel-Test Inc., Friendswood, TX) and treated with RNase-free RQ1-DNase (Promega Corp., Leiden, The Netherlands) to remove residual DNA. Differential display analysis was performed on 200 ng of RQ1-DNase-treated RNA using the RNAimage-mRNA differential display system (GenHunter Corporation, Nashville, TN), according to the manufacturer’s protocol. Reamplified differential-display polymerase chain reaction (PCR) products were sequenced directly by cycle sequencing using the Thermosequenase dye terminator cycle sequencing kit (Amersham Life Science, Cleveland, OH). Primers provided with the differential display kit were used and sequences were analyzed using the ABI 373 XL sequencer and Sequence analysis 3.3 Software (Applied Biosystems, Perkin Elmer Corp., Foster City, CA). RT-PCR was performed using GATA-3-specific primers spanning nucleotides 1227 to 1499 of the GATA-3 sequence (GenBank accession number NM_002051); (forward primer: 5′-AAG GCATCCAGACCAGAAACCG-3′; reverse primer: 5′-AGCATCGAGCAGGGCTCTAACC-3′). RT-PCR for the housekeeping gene encoding the U1 small nuclear ribonucleoprotein-specific A protein (snRNP U1A)12Snijders PJ van Duin M Walboomers JMM Steenbergen RDM Risse EKJ Helmerhorst TJM Verheijen RHM Meijer CJLM Telomerase activity exclusively in cervical carcinomas and a subset of cervical intraepithelial neoplasia grade III lesions: strong association with elevated messenger RNA levels of its catalytic subunit and high-risk human papillomavirus DNA.Cancer Res. 1998; 58: 3812-3818PubMed Google Scholar served as a reference for the semiquantitative assessment of GATA-3 mRNA levels. RT-PCR was performed as described previously12Snijders PJ van Duin M Walboomers JMM Steenbergen RDM Risse EKJ Helmerhorst TJM Verheijen RHM Meijer CJLM Telomerase activity exclusively in cervical carcinomas and a subset of cervical intraepithelial neoplasia grade III lesions: strong association with elevated messenger RNA levels of its catalytic subunit and high-risk human papillomavirus DNA.Cancer Res. 1998; 58: 3812-3818PubMed Google Scholar on 50 ng of target RNA for 28 PCR cycles. To avoid amplification of residual genomic DNA the RNAs were pretreated with RQ1-DNase (Promega) and reactions without reverse transcriptase added during cDNA synthesis were included. To quantify RNA expression, RT-PCR products were hybridized to a radiolabeled GATA-3-specific oligonucleotide probe (5′-AGACACATGTCCTCCCTGAGCCACATCTCG-3′) or an snRNP U1A-specific oligonucleotide probe,12Snijders PJ van Duin M Walboomers JMM Steenbergen RDM Risse EKJ Helmerhorst TJM Verheijen RHM Meijer CJLM Telomerase activity exclusively in cervical carcinomas and a subset of cervical intraepithelial neoplasia grade III lesions: strong association with elevated messenger RNA levels of its catalytic subunit and high-risk human papillomavirus DNA.Cancer Res. 1998; 58: 3812-3818PubMed Google Scholar respectively. Signals were quantified by Phosphorimager analysis (Molecular Dynamics, Sunnyvale, CA). mRNA expression levels were normalized to the levels measured in primary donor keratinocytes according to the following formula: intensity ratio (GATA-3/snRNP U1A) of analyzed cell culture/intensity ratio (GATA-3/snRNP U1A) of primary donor keratinocytes × 100%. Primary keratinocytes (EK00-12, 2.106 cells), SiHa (2.106 cells), and MCF-7 (0.5.106 cells) cells were lysed in 40 μl of lysis buffer (0.2 mol/L Tris-HCl, pH 6.8, 4% sodium dodecyl sulfate, 0.18% v/v glycerol, 0.02% v/v mercaptoethanol) for 5 minutes at room temperature and centrifuged at 14,000 rpm for 5 minutes. Supernatants were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to nitrocellulose. Blots were probed with murine monoclonal anti-GATA-3 antibody (HG3-31, dilution 1:500; Santa Cruz Biotechnology Inc., Santa Cruz, CA). Horseradish peroxidase-conjugated goat anti-mouse IgG1 antibody (dilution 1:5000; Southern Biotechnology Associates, Birmingham, AL) was used for visualization. Organotypic raft cultures of the primary keratinocytes and of the HPV-transformed cell lines have been described previously.13Steenbergen RDM Parker JN Isern S Snijders PJF Walboomers JMM Meijer CJLM Broker TR Chow LT Viral E6–E7 transcription in the basal layer of organotypic cultures without apparent p21cip1 protein precedes immortalization of HPV 16 and HPV 18 transfected human keratinocytes.J Virol. 1998; 72: 749-757PubMed Google Scholar Formalin-fixed, paraffin-embedded tissue specimens of normal cervix (n = 14), CIN I (n = 6), CIN II (n = 2), and CIN III (n = 9) lesions, and cervical carcinomas (n = 12), collected during the course of routine clinical practice, were obtained from women undergoing biopsy or surgery at the gynecology department of the Vrije Universiteit Medical Center in Amsterdam. Tissue specimens were previously HPV typed using the general primer GP5+/6+ PCR immunoassay (EIA) as described by Jacobs and colleagues.14Jacobs MV Snijders PJF van den Brule AJC Helmerhorst TJM Meijer CJLM Walboomers JMM A general primer GP5/GP6 mediated PCR immunoassay method for rapid detection of 14 high risk and 6 low risk human papillomavirus genotypes in cervical scrapes.J Clin Microbiol. 1997; 35: 791-795PubMed Google Scholar HPV positivity was found in 25% of normal cervical epithelium specimens analyzed (HPV type 16), in 50% of CIN I lesions (HPV types 16, 39, and 42), and in all CIN II lesions (HPV types 16, 51, and 58), CIN III lesions (HPV types 16 and 31), and cervical squamous cell carcinoma (HPV types 16, 18, and 35). Also, formalin-fixed tissue specimens of two estrogen receptor-positive breast carcinomas were subjected to immunohistochemical analysis because these have a high likelihood of overexpressing GATA-3.15Hoch RV Thompson DA Baker RJ Weigel RJ GATA-3 is expressed in association with estrogen receptor in breast cancer.Int J Cancer. 1999; 84: 122-128Crossref PubMed Scopus (165) Google Scholar Immunohistochemical staining was performed on 4-μm sections of raft cultures, and cervical and mammary tissue specimens. Endogenous peroxidase was inactivated by incubation with 0.3% H2O2 in methanol for 30 minutes. For GATA-3 detection slides were pretreated with 1 mmol/L of ethylenediaminetetraacetic acid, 0.1 mol/L Tris, pH 8.0, in an autoclave, followed by successive rinses in 0.5% Triton X-100 and 0.1 mol/L of glycine. MIB-1 detection was performed as described previously.13Steenbergen RDM Parker JN Isern S Snijders PJF Walboomers JMM Meijer CJLM Broker TR Chow LT Viral E6–E7 transcription in the basal layer of organotypic cultures without apparent p21cip1 protein precedes immortalization of HPV 16 and HPV 18 transfected human keratinocytes.J Virol. 1998; 72: 749-757PubMed Google Scholar Consecutive slides were incubated with a primary antibody against GATA-3 (HG3-31, dilution 1:200; Santa Cruz Biotechnology Inc.) and a primary antibody against the proliferation marker Ki-67/MIB-1 (MIB-1, dilution 1:100; DAKO, Glostrup, Denmark), at 4°C overnight. Antibodies were detected using the Ultravision Large Volume Detection System (Labvision Corp., Fremont, CA) and 3-amino-9-ethyl carbazole was used as a chromogen. Sections were counterstained with hematoxylin. To ensure the quality of the sections, GATA-3 staining was performed within 3 days after cutting of the sections. In addition, the majority of samples were stained with a second antibody against GATA-3 (HG3-35, dilution 1:100; Santa Cruz Biotechnology). Both GATA-3 antibodies gave very similar staining patterns in all sections analyzed, although HG3-31 staining tended to be somewhat stronger in some sections. Positive staining of T lymphocytes in the cervical stroma, as was seen in all sections, served as an internal control. Moreover, the integrity of the paraffin material was tested by staining consecutive slides for the proliferation marker Ki-67/MIB-1. All sections included in this study showed epithelial cells staining positive for Ki-67/MIB-1. Two expert pathologists performed histological examination of the stained sections. By differential display analysis a panel of RNAs isolated from FK18A and FK18B cells at the pre-immortal stage was compared with RNAs isolated from FK16A, FK16B, FK18A, and FK18B cells at the immortal stage. Eight different arbitrary primers were combined with three defined anchoring primers, resulting in 24 primer combinations. On average 65 bands were obtained for each primer combination, giving rise to an overall representation of ∼1500 mRNA species. PCR fragments were only isolated and sequenced when differential expression was observed in a major subset of pre-immortal versus immortal cells involving more than a single cell line. This does not only allow the preferred selection of genes that are more generally associated with HPV-mediated immortalization, but also reduces the chance of isolating false-positive PCR products. We isolated two PCR fragments, which displayed a reduced intensity in the immortal stages of all four cell lines (Figure 1A). On sequencing one of these fragments was found to represent a yet unidentified gene, whereas the second one was found to be identical to sequences encoding the GATA-3 transcription factor. To confirm differential expression of GATA-3 mRNA semiquantitative RT-PCR analysis was performed on RNAs isolated from the primary donor keratinocytes, different passages of pre-immortal FK16A, FK18A, and FK18B cells, and immortal cells of the four cell lines. No RNA was available from FK16B cells at the mortal stage. In addition, RNAs isolated from two cervical carcinoma cell lines, SiHa and HeLa, containing HPV 16 and HPV 18, respectively, were included in the analysis. Compared to the primary donor keratinocytes in which the percentage of GATA-3 expression was set to 100%, cells of all pre-immortal passages expressed similar levels (range, 80 to 98%) of GATA-3 mRNA. In contrast, GATA-3 expression was strongly reduced in all passages of immortal cells (range, <1 to 33%) (Figure 1B). Moreover, GATA-3 expression was markedly reduced in the cervical carcinoma cell lines SiHa and HeLa compared to the primary keratinocytes (<1%) (Figure 1B). To analyze whether the reduced mRNA expression resulted in a reduction in GATA-3 protein expression Western blot analysis was performed on total cell lysates of primary keratinocytes (EK00-12) and SiHa cells. Protein extract of the breast carcinoma cell line MCF-7, which is known to abundantly express GATA-3 and in which GATA-3 protein can specifically be detected with the monoclonal GATA-3 antibody HG3-31,15Hoch RV Thompson DA Baker RJ Weigel RJ GATA-3 is expressed in association with estrogen receptor in breast cancer.Int J Cancer. 1999; 84: 122-128Crossref PubMed Scopus (165) Google Scholar was included as a positive control. In primary keratinocytes GATA-3 protein was detected, although at a much lower level as in MCF 7 cells. In contrast, GATA-3 protein expression was undetectable in SiHa cells (Figure 1C). Analysis of GATA-3 protein expression in three-dimensional organotypic raft cultures of the primary donor keratinocytes, immortal FK18B cells, and SiHa cells by immunohistochemistry showed an almost complete absence of GATA-3 expression in immortal FK18B and SiHa cells. In contrast, primary keratinocytes showed clear GATA-3 staining (Figure 2). Taken together, these data show that both GATA-3 mRNA and protein expression is markedly reduced in HPV-immortalized cells and cervical cancer cells compared to primary keratinocytes and pre-immortal HPV-transformed cells. We subsequently tested whether differential expression of GATA-3 also occurs in cervical lesions. To this end GATA-3 expression was analyzed by immunohistochemistry in 14 normal cervical squamous epithelial specimens, 17 CIN lesions (6 CIN I, 2 CIN II, and 9 CIN III) and 12 cervical squamous cell carcinomas. In normal cervical epithelia nuclear GATA-3 expression could be clearly detected in all tissue samples analyzed. Staining was most pronounced in the lower half of the squamous epithelium and was decreased in apical, more differentiated, epithelial layers. No staining was seen in columnar epithelial cells of the endocervix. GATA-3 immunostaining was also observed in all CIN I lesions (n = 6) both in basal and suprabasal cell layers, although two of these lesions revealed somewhat weaker staining compared to normal epithelium. Both CIN II lesions analyzed showed a weak GATA-3 staining in basal and suprabasal layers. Interestingly, one of the nine CIN III lesions was completely negative for GATA-3, whereas normal epithelium in the same section and lymphocytes underneath the CIN lesion were strongly positive. Another CIN III lesion showed focal staining for GATA-3 in some areas and four CIN III lesions showed sporadically weak GATA-3-positive cells. The three remaining CIN III lesions showed clear GATA-3 staining; one in the basal layer, one in the lower half of the epithelium, and one throughout the epithelium. Of 12 carcinomas analyzed, 8 revealed complete absence of GATA-3 staining in the tumor cells, whereas in surrounding stroma clear GATA-3-positive lymphocytes could be observed. Two other carcinomas showed focal GATA-3 expression with certain tumor areas staining very weakly positive whereas other areas were completely negative. In all these cases clear positive lymphocytes were detected in the surrounding stroma. The two remaining cervical carcinomas showed clear positive GATA-3 staining in the tumor areas. Representative examples of GATA-3 immunostaining on tissue specimens are shown in Figure 3. Both estrogen receptor-positive breast carcinomas analyzed showed GATA-3 immunopositivity with staining intensities that were markedly higher than that observed in normal cervical epithelium (data not shown). This is in line with the different expression levels seen in the Western blot for primary keratinocytes compared to the estrogen receptor-positive MCF-7 cell line. In this study we showed that HPV-mediated immortalization in vitro is associated with a down-regulation of the transcription factor GATA-3. The reduction in GATA-3 mRNA expression during immortalization seemed to be a common feature for all four different HPV-transformed cell lines and the cervical cancer cell lines analyzed. Further Western blot analysis and immunohistochemical analysis of organotypic raft cultures of the cell lines revealed that reduction in GATA-3 mRNA expression is translated into a clearly reduced protein expression in HPV-immortalized cells and cervical carcinoma cells compared to primary keratinocytes. In addition, immunohistochemical analysis of cervical tissue specimens revealed complete loss of GATA-3 staining in 11% of CIN III lesions and 67% of cervical carcinomas. These results indicate that loss of GATA-3 expression is not only associated with immortalization in vitro, but is also with cervical carcinogenesis in vivo. In addition, these data suggest that complete loss of GATA-3 expression occurs rather late in the CIN to cervical carcinoma sequence. To our knowledge this is the first study showing expression of GATA-3 in primary human keratinocytes and normal squamous epithelium. GATA-3 was originally identified as a transcription factor that binds to and activates the T-cell receptor gene enhancer.16Ho I-C Vorhees P Marin N Karpinski-Oakley B Tsai S-F Orkin SH Leiden JM Human GATA-3: a lineage-restricted transcription factor that regulates the expression of the T cell receptor_? gene.EMBO J. 1991; 10: 1187-1192Crossref PubMed Scopus (259) Google Scholar GATA-3 belongs to the GATA family of transcription factors, which bind to the consensus sequence (A/T)GATA(A/G) and to the related motifs CGATGG and (T/A)GAT(T/A)(A/G).17Ko LJ Engel JD DNA-binding specificities of GATA transcription factor family.Mol Cell Biol. 1993; 13: 4011-4022Crossref PubMed Scopus (502) Google Scholar GATA-3 gene knockout studies revealed a critical role for GATA-3 in embryogenesis.18Pandolfi P Roth M Karis A Leonard M Dzierzak E Grosveld F Engel JD Lindenbaum MH Targeted disruption of the GATA-3 gene causes severe abnormalities in the nervous system and in the liver haematopoiesis.Nat Genet. 1995; 11: 40-44Crossref PubMed Scopus (495) Google Scholar In adults, GATA-3 has been shown to act as a transcriptional activator in T-cell differentiation,16Ho I-C Vorhees P Marin N Karpinski-Oakley B Tsai S-F Orkin SH Leiden JM Human GATA-3: a lineage-restricted transcription factor that regulates the expression of the T cell receptor_? gene.EMBO J. 1991; 10: 1187-1192Crossref PubMed Scopus (259) Google Scholar whereas it has been described to act as a transcriptional repressor during adipocyte differentiation.19Tong Q Dalgin G Xu H Ting C-N Leiden JM Hotamisligil GS Function of GATA transcription factors in preadipocyte-adipocyte transition.Science. 2000; 290: 134-138Crossref PubMed Scopus (388) Google Scholar Moreover, GATA-3 haplo-insufficiency was found to be involved in the etiology of human malformations, ie, the hypoparathyroidism, sensorineural deafness, renal anomaly syndrome (HDR).20van Esch H Groenen P Nesbit MA Schuffenhauer S Lichtner P Vanderlinden G Harding B Beetz R Bilous RW Holdaway I Shaw NJ Fryns J-P Van de Ven W Thakker RV Devriendt K GATA3 haplo-insuficiency causes human HDR syndrome.Nature. 2000; 406: 419-422Crossref PubMed Scopus (420) Google Scholar The GATA-3 gene is located at 10p15, a region, which is more than incidentally altered in HPV-immortalized cells and in cervical carcinomas.4Steenbergen RDM Walboomers JMM Meijer CJLM van der Raaij-Helmer Parker JN Chow LT Broker TR Snijders PJF Transition of human papillomavirus type 16 and 18 transfected human foreskin keratinocytes towards immortality: activation of telomerase and allele losses at 3p, 10p, 11q and/or 18q.Oncogene. 1996; 13: 1249-1257PubMed Google Scholar, 21Reznikoff CA Belair C Savelieva E Zhai Y Pfeifer K Yeager T Thompson KJ De Vries S Bindley C Newton MA Sekhon G Waldman F Long-term genome stability and minimal genotypic and phenotypic alterations in HPV 16 E7-, but not E6-, immortalized human uroepithelial cells.Genes Dev. 1994; 8: 2227-2240Crossref PubMed Scopus (131) Google Scholar, 22Solinas-Toldo S Dürst M Lichter P Specific chromosomal imbalances in human papillomavirus-transfected cells during progression toward immortality.Proc Natl Acad Sci USA. 1997; 94: 3854-3859Crossref PubMed Scopus (79) Google Scholar, 23Savelieva E Belair CD Newton MA DeVries S Gray JW Waldman F Reznikoff CA 20q gain associates with immortalization: 20q13.2 amplification correlates with genome instability in human papillomavirus 16 E7 transformed human uroepithelial cells.Oncogene. 1997; 14: 551-560Crossref PubMed Scopus (123) Google Scholar, 24Steenbergen RDM Hermsen MAJA Walboomers JMM Meijer GA Baak JPA Meijer CJLM Snijders PJF Non-random allelic losses at 3p, 11p and 13q during HPV-mediated immortalization and concomitant loss of terminal differentiation of human keratinocytes.Int J Cancer. 1998; 76: 412-417Crossref PubMed Scopus (37) Google Scholar, 25Stanley M Sarkar S Genetic changes in cervical carcinoma.Papillomavirus Rep. 1994; 5: 141-147Google Scholar, 26Cottage A Dowen S Roberts I Pett M Coleman N Stanley M Early genetic events in HPV immortalised keratinocytes.Genes Chromosom Cancer. 2001; 30: 72-79Crossref PubMed Scopus (34) Google Scholar In addition, putative senescence and telomerase repressor loci have recently been identified at 10p14-p15 and 10p15, respectively.27Poignee M Backsch C Beer K Jansen L Wagenbach N Stanbridge EJ Kirchmayr R Schneider A Durst M Evidence for a putative senescence gene locus within the chromosomal region 10p14–p15.Cancer Res. 2001; 61: 7118-7121PubMed Google Scholar, 28Nishimoto A Miura N Horikawa I Kugoh H Murakami Y Hirohashi S Kawasaki H Gazdar AF Shay JW Barrett JC Oshimura M Functional evidence for a telomerase repressor gene on human chromosome 10p15.1.Oncogene. 2001; 20: 828-835Crossref PubMed Scopus (46) Google Scholar Therefore, further functional studies are warranted to find out whether GATA-3 deregulation is involved in HPV-mediated immortalization and cervical carcinogenesis. Although this is the first demonstration of a potential role for GATA-3 in the development of cervical cancer, recent reports have described altered expression of GATA transcription factors in esophageal carcinoma cells,29Shiga K Shiga C Sasano H Miyazaki S Yamamoto T Yamamoto M Hayashi N Nishihira T Mori S Expression of c-erbB-2 in human esophageal carcinoma cells: overexpression correlated with gene amplification or with GATA-3 transcription factor expression.Anticancer Res. 1993; 13: 1293-1302PubMed Google Scholar, 30Lin L Aggarwal S Glover TW Orringer MB Hanash S Beer DG A minimal critical region of the 8p22–23 amplicon in esophageal adenocarcinomas defined using sequence tagged site-amplification mapping and quantitative polymerase chain reaction includes the GATA-4 gene.Cancer Res. 2000; 60: 1341-1347PubMed Google Scholar breast carcinomas,15Hoch RV Thompson DA Baker RJ Weigel RJ GATA-3 is expressed in association with estrogen receptor in breast cancer.Int J Cancer. 1999; 84: 122-128Crossref PubMed Scopus (165) Google Scholar and gastric carcinoma cell lines.31Bai Y-Q Akiyama Y Nagasaki H Yagi OK Kikuchi Y Saito N Takeshita K Iwai T Yuasa Y Distinct expression of CDX2 and GATA4/5 development-related genes, in human gastric cancer cell lines.Mol Carcinog. 2000; 28: 184-188Crossref PubMed Scopus (52) Google Scholar In conclusion, down-regulation of GATA-3 was correlated with HPV-mediated immortalization in vitro and advanced (pre)malignant cervical disease. Therefore, it may provide a marker for progressive CIN disease. We thank Elly Fieret and Monique Egging for excellent technical assistance with the immunohistochemical analysis.
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